bacteria identification & classification Flashcards
what are the 3 steps for identifying bacteria for the bacteriology laboratory?
- correct sampling procedures & provision of adequate history
- transport to lab to ensure viability of suspect organism & minimal contamination
- growth, identification & antibiotic sensitivity testing
what does the correct sampling procedure step for identifying bacteria involve?
- obtaining the specimen aseptically from the disease site
- collecting the specimen at the correct time during disease process (acute stage of infection)
- if collecting sample from a dead animal - collect sample before post-mortem changes occur & from site most likely to yield pathogens
what information is collected for the provision of an adequate history?
collection of a full clinical history
- age, sex, species, number of animals affected (if production animal)
- treatment administered
- tentative clinical diagnosis
what does the transport to the lab step involve?
- involves ensuring viability of suspect organism & minimising contamination
- transport media should be a semi-solid agar containing salts & buffers
- keep sample at 4*C
- body fluids other than urine should be held at room temperature
- submitting sample in leak proof containers
- simple should be labelled & delivered to lab within 48hrs
once sample is transported to the lab, how do you choose what media to grow bacteria on?
if suspecting:
- mucosal infection - use blood agar & MacConkey agar
- anaerobic bacteria from body fluid sample - add a second agar plate incubated anaerobically
- fastidious organisms - use chocolates or specialised agar
- fungal pathogens - add Sabouraud’s agar plate plus chloramphenicol
what does the growth, identification & antibiotic sensitivity testing step involve for identifying bacteria?
- choose media, plate out & incubate
- initial smear on gram stain is the first step in identification of a bacterial organism - guides choice of initial therapy
- examine plates every 24 hours to look at colony morphology on solid media
- obtain a pure culture - as bacterial identification can only be performed on pure cultures of bacteria (all descendants from one bacterial cell)
- perform a minimum number of tests to identify the isolate to a species level
- tests mostly biochemical, some microscopic, immunological or molecular - determine antimicrobial sensitivity profile
how is a pure culture obtained?
- spread bacteria on an agar plate to achieve individual bacterial colonies from a single cell
- a large amount of organism in PURE culture is required to do more tests
when is solid media chosen and when is broth chosen as the media to grow organisms on?
- generally both liquid & solid media are innoculated for microbe isolation
- solid media - grows individual colonies, allowing quantification of bacteria & differentiation of normal flora from potential pathogen
- broth - allows recovery of a small number of organisms or those that are more fastidious
define a bacterial strain
- a line of bacteria descended from a single ancestor through various subcultures
define an isolate?
- is each primary culture isolated from a natural source
what do biochemical tests detect when we use them to identify bacteria?
- detect enzyme activity
- detect end products to identify substrate utilisation
- eg rapid kits, oxidase test, catalase test
what do immunological tests detect when we use them to identify bacteria?
- detect antigen-antibody reactions (agglutination reaction)
- clumping indicates presence of antigen related to a specific species of bacteria
- used to identify bacteria sterotypes/serovars
define serotypes
- are groups of microorganisms grouped together based in their cell surface antigens
why would a molecular test be chosen as the method to identify bacteria?
can be used to identify bacteria that:
- cannot be cultured
- are slow or difficult to grow
- when rapid diagnosis is important
- can be used to measure antimicrobial resistance
what are the 3 approaches of molecular tests that can be used to identify bacteria?
- immunological - includes
- serology of surface antigens
- or detecting other markers - chemical/physical
- includes simple indirect tests
- or MALDI-TOF - genetic
- PCR
- genome sequencing
- molecular typing
what can PCR be used for?
- identifying bacteria to a species level
- identifying strain
- characterising bacteria (eg virulence genes)
- real time PCR gives instant results & can estimate initial bacterial load
what is MALDI-TOF used for?
Matrix Associated Laser Depolarisation/Ionisation-Time of Flight
- MALDI-TOF is a form of matrix spectrometry that identifies bacterial species
- high capital cost but low operation cost
- only as good as reference database
what is molecular typing used for?
finger-printing of bacteria using genomics
- determining matching strains for outbreak investigations
- determining phylogenetic relationships
- identifying sub-species & sub-types
what is whole genome sequencing detect during bacteria identification?
- can detect single nucleotide polymorphisms
- allows better comparisons between labs
- provides evolutionary info
what are the limitations to molecular methods that can be used to identify bacteria?
- cost & availability
- design & validation of the procedure
- quality of data base library
- accuracy & viability of interpretation
- some level of culture is often still required to get enough info
- no isolate in sample
how are bacteria classified?
- bacteria classification is based on a set of morphological, biochemical & genetic characteristics
what is the gold standard in bacterial taxonomy?
sequencing of 16S ribosomal RNA molecules
- provides genetic similarity above species level
- improves identification of some bacterial pathogens
- but increasingly being replaced by whole genome sequencing
how are bacteria named?
- bacteria are given a genus name & a species name
- species levels is the most important rank in taxonomic hierarchy
- 16S RNA sequence must be >98% identical to belong in same species
- I6S RNA sequences must to be >95% identical to belong in same genus
